The Study Of Polyhydroxyalkanoate Production From Lignin By Pseudomonas Putida NX-1 Based On The Genome Information

Lignocellulose,the most abundant renewable organic polymer on the planet,is composed of cellulose,hemicellulose,and lignin.In current status,lignocellulose is one of the potential candidates to replace fossil resources.With the development of lignocellulose biorefienry,cellulose and hemicellulose can be converted into fuels and chemicals through a series of processes,mainly including pretreatment,enzymatic hydrolysis and fermentation.However,the residual lignin has not yet been effectively utilized in current biorefinery concept.As a major by-product of lignocellulose biorefinery field,the effective use of lignin is of great significance to both economics and carbon utilization efficiency of biorefinery.In nature,the degradation and mineralization of lignin is an important component of the whole carbon cycle.Some natural microorganisms can depolymerize high-molecular-weight lignin,and utilize the generated lowmolecular-weight lignin for their growth,metabolism,as well as product synthesis,which provides a basis for the biological valorization of lignin.Based on previous studies,most of the lignin degrading strains are fungi,mainly including white rot,brown rot and soft rot,among which,Phanerochaete chrysosporium has become a model microorganism for the study of lignin biodegradation due to its outstanding lignin degradation ability.Compared with fungi,some bacteria strains have some advantages in industrial production,e.g.faster cell growth,and more convenient genetic operations.Moreover,many bacteira strains exhibit good tolerance to extreme p H and temperature,as well as high lignin concentration.However,less studies on lignin degradation bacteria and on the ligninolytic enzymes from bacteria have been reported compared with fungi,which limit the application of bacteria in lignin valorization.In a recent study,some lignin biodegrading bacteria strains were screened by our group,among which Pseudomonas putida NX-1 exhibited the best lignin-degrading ability.Based on the above background,some works were performed as present below:1.The genome of P.putida NX-1(Gene bank:NZ＿CP030750.1)was sequenced by Single Molecule,Real-Time(SMRT)technology.The annotated information suggested that there are abundant enzymes and pathways involving in the depolymerization of high-molecularweight lignin,such as laccase,peroxidase,manganese peroxidase,and dye-decolorizing peroxidase.In addition,some enzymes and biodegradation pathways related to the assimilation of low-molecular-weight lignin,such as of vanillin,p-hydroxybenzoic acid,as well as protocatechuic acid,were also mined from P.putida NX-1 based on the genome information.2.Among the predicated enzymes related to lignin biodegradation,a recent reported dyedecolorizing peroxidase raise our attention because it commonly plays an important role in lignin biodegradation in some reported bacteria strains.To confirm the function of this dyedecolorizing peroxidase,it was heterologously expressed in Escherichia coli and purified by a Ni-sepharose.Then,some of the enzymes properties were investigated,which indicated this enzyme can use some ligninmimicking compounds as substrates,including Reactive Blue 19,vanillic acid,2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),and syringic acid.3.In addition to pahthways involving in lignin biodegradation,the genome information also suggested that there is a complete polyhydroxyalkanoate(PHA)synthetic pathway in P.putida NX-1.Moreover,these pathways of lignin biodegradation and p HA synthesis are connected with Acetyl Coenzyme A as a key intermediate in P.putida NX-1,which suggested that P.putida NX-1 possesses the capability of producing PHA using lignin as substrate.Then,the PHA biosynthesis capability of P.putida NX-1 was confirmed in a high carbon-nitrogen ratio condition with lignin as the sole carbon source.Furthermore,the monomer compositions,the molecular weights,and thermal properties of PHA produced from glucose and lignin derived aromatics were comprehensively compared,and the results indicated that physical properties of PHA prepared from glucose and lignin-derived aromatics are similar,which suggested that lignin can be an alternative substitute for glucose for polyhydroxyalkanoate production.